The present invention relates to a method and a stable reagent for the chemical diagnosis of a pathogenic bacterial infection in humans. More particularly, the present invention relates to the production of a stable reagent which permits qualitative determination of streptococci infections by unaided visual determination.
When a human or other animal is exposed to foreign proteins of antigenic character, an immunologic response will often occur. One aspect of this response is the presence in the animal's blood of water-soluble antibodies capable of specifically recognizing and binding the foreign protein.
During a bacterial infection in an animal, the invading organisms may excrete a great number of these foreign proteins. Streptococci bacteria have been shown to excrete over 20 exocellular toxins, one of which is a cytolytic protein known as streptolysin-O.
Streptolysin-O is an extremely destructive protein, one of its prime functions being the hemolysis of red blood cells. Fortunately, streptolysin-O is also antigenic and the animal can respond immunologically with the appropriate antibodies (anti-streptolysin) which can inhibit lysogenesis by binding onto the streptolysin-O.
The amount of anti-streptolysin present in serum, called the "titer" is an important tool to clinicians, not only for the diagnosis of streptococci infection, but also for its relationship with rheumatic fever, acute gloumerulonephritis, rheumatoid arthritis and erythema nodosum. To determine this titer, some technicians have utilized the inhibitory properties of anti-streptolysin in conjunction with the lytic properties of streptolysin-O on red blood cells. In this respect, the study done by Rammelkamp [Am. J. Med. 10:673, 1951] teaches one such method.
The most commonly used method for determining the concentration of streptolysin-O antibodies is to perform a hemolytic inhibition test. One of the activities of the streptolysin-O is its ability to hemolyze human red blood cells and blood cells of other animal species. In the presence of anti-streptolysin in the patient's serum, the hemolysis is inhibited. Thus, an increased concentration of anti-streptolysin present in the serum will be reflected in greater inhibition. Quantitative expression is measured by an elaborate dilution scheme of the patient's serum.
The isolation of streptolysin-O from the culture medium is a long and tedious process. In addition, after several days of handling in an aerobic atmosphere, the toxin is in an oxidized form. For hemolytic activity, streptolysin-O must be in a reduced state. This is usually accomplished by the addition of cysteine, 2-mercaptoethanol or other thiols as reducing agents. Even with the addition of any of these compounds, the hemolytic potency of the toxin is short lived. Commercially available streptolysin-O reagents are usually reduced, freeze dried, and sealed under an inert gas atmosphere. Under reconstitution, the reduced material must be used within thirty minutes.
Although this test procedure is widely used, it is tedious and necessitates maintaining a constant supply of viable red blood cells. Also, because streptolysin-O loses potency when oxidized, the procedure must be done quickly or in the presence of reducing agents which may adversely affect the analysis.
From the literature, it is apparent that streptolysin-O and its activity will form precipitins without the necessity of activating the oxidized toxin. Thus, the indication is that while the hemolytic activity is dependent on the toxin being in a reduced form, the precipitin formation is independent of its redox state.
Antigen-antibody reactions may manifest themselves under various conditions. In soluble forms, the antigen and antibodies will combine to form loose aggregates, which continue to a lattice build-up to become visible precipitates. If this soluble antigen-antibody reaction should take place in a semi-solid medium such as agar, a macroscopically visible precipitin line will result.
Attempts to simplify the foregoing test procedure have been based on the well-known antibody agglutination process and the fact that anti-streptolysin recognition of streptolysin-O is independent of the toxin's redox state. In addition, due to the importance of qualitative determination of anti-streptolysin titers, much research has been directed towards rendering the agglutination product macroscopically visible.
The conversion of a "precipitin" reaction to an "agglutination" reaction has occupied the time and efforts of clinical immunochemists throughout the world for the past three decades. Adsorption of a reactant to a carrier particle serves to demonstrate the presence of either the antigen or antibody within a relatively short length of time, and secondly, the macroscopic visualization of the antigen-antibody reaction does not require the need for elaborate or sophisticated equipment. For these reasons, simple agglutination reactions have enjoyed wide popularity in laboratory use of immunodiagnosis of disease states. Various carrier particles such as erythrocytes, bentonite, collodium, quartz, synthetic resins and latex particles have been employed as the serologic carrier of one of the reactants.
In instances of antigen-antibody reaction, where the reactant is in a particulate form, visible agglutination of the particles will demonstrate the antibody-antigen reaction.
Technical difficulties, availability, variation in sizes and compositions have eliminated many of the aforementioned carriers as reactant particles. However, commercially available uniform size polystyrene latex has enjoyed immense popularity as a serologic carrier.
Various techniques for forming visible agglutination products in general are known in the art and good results have been obtained by adsorbing either the antigen or the antibody to a carrier. In this regard, U.S. Pat. No. 3,088,875 teaches the use of polymerized styrene latex particles as carriers.
Polystyrene latex particles are usually hydrophobic and negatively charged, and will non-specifically adsorb many proteinaceous materials. However, because the adsorption is often not permanent, a number of procedures have been developed to stabilize the protein-latex complex. These methods usually require some modification of the protein and the addition of extraneous high molecular weight materials. A representative sampling of these procedures can be found in U.S. Pat. Nos. 3,658,982 and 3,992,517, and German Pat. No. 1,914,081.
These polystyrene particles are usually prepared by initiating the polymerization of styrene monomers with potassium persulfate in the presence of the emulsifier sodium laurel sulfate. In preparation of particles through emulsion polymerization the stability of the polystyrene particles is due in part to the charged surface groups originated from the initiator through surface sulfate groups. However, the emulsifier also contributes to the stability of the particles by imparting an electrical negative charge. The greater the quantity of emulsifier the greater the negative charge on the latex particle surface. A double layer of ions and counter ions is usually responsible for the stability of the particles. Divalent and trivalent cations as well as high ionic strength buffers may cause destabilization of the polystyrene latex colloid.
A review of the art reveals that IgG molecules containing antibody activity are usually non-specifically adsorbed onto the polystyrene latex particles which then serve as a reagent. Heat and other methods of denaturation will tend to "stabilize" the reagent. These antibodies, usually composed of negatively charged IgG molecules, will be adsorbed onto the negatively charged latex particles due to the influence of the non-ionic van der Waals forces and the hydrophobic bond effects.
When streptolysin-O is combined with polystyrene latex particles by alteration of the protein, either through partial degradation or by the addition of stabilizing materials, the modification may cause a loss of some antigenic determinants and a decrease in assay sensitivity. Furthermore, the extraneous materials utilized may interfere with the antibody-antigen interaction or the agglutination reaction, resulting in false results and incorrect clinical diagnosis.
Thus, those skilled in the art have recognized a significant need for a stable streptolysin-O-latex complex in which the protein retains significant preadsorption characteristics and wherein the entire complex is free from potentially contaminating intermediaries. The present invention fulfills this need.